Fig. 6. The phenotypic effects induced by ectopic Slug expression in MDCK cells are associated with a full repression of E-cadherin expression and are independent of endogenous Snail expression. (A) Western blot analysis of whole cell extracts of the indicated proteins in mock- and Slug-transfected clones. E-cad, E-cadherin; Plako, plakoglobin; ß-cat, ß-catenin; VN, vimentin. Detection of -tubulin (-tub) levels was used as a loading control. (B) The presence of canine E-cadherin, mouse Slug and canine Snail transcripts in mock- and Slug-transfected clones was analyzed by RT-PCR. The expression of GAPDH was analyzed in the same samples as a control for the amount of cDNA present in each sample. The —RT lane shows the results of amplification in the absence of template. (C) Inmunofluorescence analysis for Slug expression in mock (a) and Slu 3 clone (b,c). Ectopic expression of the Slug protein was observed in the nuclei of the Slug-transfected cells (arrows in b and c). Bar, 20 µm. (D) The activity of the mSnail promoter in the Slug-transfectants corresponds to the endogenous Snail mRNA levels. The indicated cell lines were transiently transfected with the wild-type mouse Snail promoter construct (white bars) or with the mutant E-box (at -221) construct (grey bars) fused to a luciferase reporter gene. Luciferase and renilla activities were determined 24 hours after transfection. The activity of the promoter is expressed relative to that obtained in the mock-transfected cells with the wild-type construct. Results represent the mean±s.d. of at least two independent experiments.

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